The instant application should be granted the priority dates of Aug. 23, 2010, the filing date of the corresponding German patent application 102010035104.0, as well as Aug. 23, 2011, the filing date of the International patent application PCT/EP2011/004227.
The invention relates to a method as well as an apparatus for automated examination of biological material with a microscope, on the cross table of which the biological material is arranged between an object slide or an analysis plate and at least one cover, with at least one light source for exposure of the biological material as well as with an image recording unit recording at least one image of the biological material enlarged by means of the object lens of the microscope and transferring it to an evaluation unit. The technical solution described provides labeling, which is detectable by a device for automatic focusing of the biological material, wherein the focusing can be executed by targeted movement of the cross table taking the detection of the labeling as the basis.
In medical diagnostics, fluorescence microscopy represents a standard method for the examination of patient samples. In that, in this area, too, an attempt in respect of the further development of this technology consists in increasing the degree of automation in order to thus minimize the error probability and to improve the economic efficiency of microscopy.
In order to achieve an increase in the degree of automation at the laboratory, it is conceivable, on the one hand, to further automate the processing of the samples to be examined, in particular the dilution as well as the incubation steps, or, on the other hand, to design the process of the predominantly visual evaluation of the processed samples more effectively. The invention explained in more detail in the following lies in the field of sample evaluation or diagnostics, respectively, wherein also in respect of fluorescence microscopy it is already known in principle to generate digital image data of the samples to be examined and to transfer these to a data processing unit for the preparation of diagnostic findings, on which unit a special laboratory software is installed, and to evaluate the data in a computer-aided fashion.
With fluorescence microscopy at least partially automated in such manner, the generation and selection of high quality images is of outstanding significance, above all in order to be able to guarantee constantly high quality in the preparation of a laboratory result. A substantial quality feature of the recorded images ultimately is their sharpness, so that focusing of the biological material to be examined is of particular significance. For focusing of the biological material or the substrates, respectively, to be examined, usually a multitude of images is recorded at different levels, of which images those with low quality are discarded during the electronic evaluation.
In respect of automated focusing for microscopes, different technical solutions are known. In that, active autofocus systems, which are normally used in reflected-light microscopes, are characterized by the fact that using an auxiliary light source, a light spot or a marking is projected onto the surface of the sample to be examined or a cover slip covering the sample, then shape, position or size of the light spot are evaluated, and, on the basis of the evaluation, focusing on the sample or cover slip surface takes place. An active autofocus system designed in such manner is known, for example, from DE 34 46 727 C2, and is to enable fast automatic focusing.
In transmitted-light microscopes, passive autofocus systems are normally used instead of active ones. Passive autofocus systems, as, e.g., known from DE 34 39 304 C2, determine the focus level on the basis of a comparison of the recorded image data, i.e. the level, at which the sharpest image was recorded. Frequently, however, such systems are comparatively slow, since a multitude of search runs must be undertaken in order to achieve a satisfactory result.
A further special active autofocus system is known from DE 10 2010 035 104. In this printed publication, an apparatus for automatic focusing of weakly luminescent substrates is described, which is to guarantee fast image focusing. The comparatively fast focusing, on the one hand, is to increase the effectiveness of automatic microscopy and, on the other hand, minimize fading of the fluorescent dyes during focusing. The technical solution described is characterized by the fact that first, several recordings are undertaken in a transmitted-light mode, and therefrom the sharpest image is determined using known evaluation methods. Following switching-off of the transmitted-light source, the cross table of the microscope is moved to the focus level determined in the transmitted-light mode. In order to generate a fluorescence image, an excitation light source is then activated. Since, however, the fluorescence can vary at various levels in respect of the thickness of the substrate, it is necessary to still adjust the focus level in the fluorescence mode compared to the focus level determined in the transmitted-light mode on a regular basis. For determination of the exact focus level, however, due to the pre-focusing previously undertaken in the transmitted-light mode, only few images must be included in a comparatively small search area. The fluorescence images ultimately lying at the exact focus level are recorded by means of a digital camera and transferred to a data processing unit for the further preparation of diagnostic findings.
One objective of the further development of autofocus systems in fluorescence microscopy always is to minimize the time required for focusing or the number of images recorded during focusing and unusable for later examination of the substrate, respectively. For that, a further method for automatic focusing of a substrate in fluorescence microscopy is known from DE 101 00 247 A1, using which the object lens focus can be determined in a highly exact manner and still within a comparatively short period. In this case, an interference microscope is described, in which a surface of the object slide unit is provided with a coating detectable by light microscopy. The reflected light is detected by a detector, and on the basis of the detection signals, the phase position in the object area of the interference microscope is concluded. In this manner, it is finally possible to adjust the interference microscope in a suitable manner.
The microscopes with automatic or at least partially automated focusing previously described are frequently used to make special proteins visible by means of antibodies. In this manner, it is determined in which tissue a special protein exists and in which compartment of a cell it is located. Frequently, fixed tissue consisting of tissue sections is used for this antibody staining. Such tissue sections are generated by first preparing a frozen section of a healthy tissue and applying it to the surface of a glass slide, in particular an object slide or a cover slip. Subsequently, the tissue is thawed and dried. Normally, the tissue sections for the known test systems are attached to standard object slides, wherein for each tissue section, a separate standard object slide is used.
Beside the previously described possibility for examination of a patient sample using a tissue section arranged on a standard object slide, the so-called biochip technology of the company Euroimmun AG is known. Contrary to the conventional technology of indirect immunofluorescence, for which a tissue section is always attached to a standard object slide provided for the examination, this technology, due to a miniaturization of the samples to be examined, offers the possibility to standardize the determination of auto-antibodies and infection antibodies. In this manner, the work at the laboratory is facilitated and becomes more efficient.
The biochips of the company Euroimmun are comparatively small slides with biological material. In that, the effect is utilized, that very small tissue sections can be prepared and used for examination, provided that these are applied to a substrate carrying the section, and together with this substrate they are attached to an object slide or an analysis plate. While a standard cover slip is an about 100 μm to 200 μm thin, rectangular or round glass platelet, which usually has an area of 18×18 mm2, biochips are cover slip fragments coated with suitable biological material, which thus have a much smaller surface. Therefore, compared to standard cover slips, biochips are on the one hand characterized by a distinctly lower space requirement, and on the other hand, the amount of tissue required is considerably reduced.
For the manufacture of a biochip, first a frozen section of the required tissue is likewise applied to a standard cover slip, then partially thawed and dried. Subsequently, the cover slip with the tissue is fragmented by producing score lines in the area of the tissue section using a diamond tip or a laser, along which the coated cover slip is broken and thus divided into segments. In order to enable an improved, in particular more effective examination, the biochips are applied to suitable reaction fields of an object slide. In this connection, it is conceivable to provide a multitude of reaction fields for respective biochips on one object slide, wherein in one reaction field, more than one biochip, preferably with different tissues, may also be provided. In order to design the examination of a patient sample particularly effective, suitable BIOCHIP-Mosaiks™ are formed by providing a multitude of reaction fields with respective biochips on one object slide.
No matter, whether a tissue section located on a standard cover slip or a biochip or special BIOCHIP-Mosaiks™, respectively, is used for the examination of a patient sample, the basic course of sample processing, in particular dilution of the patient serum as well as incubation of the tissue section, as well as of the visual examination using a microscope is the same.
During processing of the sample, the tissue is incubated with diluted patient serum, which is to be examined for the presence of special antibodies, as well as a conjugate, which normally includes antibodies originating from an animal, which are marked with a fluorescent substance and directed against the antibodies suspected in the patient serum. If the patient serum has antibodies against the antigens of the tissue section, these antibodies bind to the tissue section and the fluorescently marked secondary antibodies bind to the human antibodies attached to the tissue section. Ultimately, the fluorescent dye bound to the respective tissue structures can be detected using a fluorescence microscope.
Prior to using the microscope, the incubated tissue section is coated with a mounting medium, like e.g. ph-buffered glycerin, and covered with a cover slip. The cover slip is arranged such that though it is located at a secure distance from the surface of the tissue section, viewing it with the microscope is still possible without any problem.
As already indicated, the objective for the further development of automatic apparatuses for the examination of patient samples principally consists in designing any procedural steps from receipt of the sample up to preparation of a laboratory result as efficient as possible, simultaneously eliminating possible sources of error as far as possible. Taking this general attempt of making laboratory work more effective and even more reliable as the basis, the present invention is based on the special object to further develop the technical solutions for automatic microscopy of biological material known from the state of the art such that automation of the microscopy of an object slide with biochips located thereon, in particular with BIOCHIP-Mosaiks, is possible in an advantageous manner. Above all, the process of focusing of the biological material arranged on the biochip(s) is to be realizable with comparatively simple means and simultaneously take place quickly as well as with high quality. The technical solution to be stated is to be easy to integrate into the known systems for automatic microscopy of biological material and represent an aid for the personnel working at the laboratory. A further object, on which the present invention is based, consists in minimizing the number of recordings made during focusing and not required later for image evaluation.